U.S. patent application number 14/460732 was filed with the patent office on 2015-06-18 for method and compositions for treating cancer using probiotics.
The applicant listed for this patent is THE UNIVERSITY OF HONG KONG. Invention is credited to Hani El-Nezamy, Jiandong Huang, Nikki Pui-Yue Lee, Cecilia Ying Ju Sung.
Application Number | 20150164964 14/460732 |
Document ID | / |
Family ID | 52468071 |
Filed Date | 2015-06-18 |
United States Patent
Application |
20150164964 |
Kind Code |
A1 |
El-Nezamy; Hani ; et
al. |
June 18, 2015 |
Method and Compositions for Treating Cancer Using Probiotics
Abstract
The subject invention pertains to probiotic compositions and
methods of treating hepatocellular carcinoma (HCC) using the
probiotic compositions. The probiotic compositions can inhibit
growth of HCC. The probiotic compositions can reduce the risk of
HCC. The probiotic compositions include a specific combination of
three bacterial compositions: (a) heat-inactivated Lactobacillus
acidophilus (Moro) Hansen and Mocqu (ATCC 53103), (b) viable
Escherichia coli Nissle 1917, and (c) heat-inactivated VSL#3.RTM..
This combination of bacteria is effective to reduce hepatocellular
carcinoma (HCC) growth compared to control growth.
Inventors: |
El-Nezamy; Hani; (Hong Kong,
CN) ; Lee; Nikki Pui-Yue; (Hong Kong, CN) ;
Sung; Cecilia Ying Ju; (Hong Kong, CN) ; Huang;
Jiandong; (Hong Kong, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE UNIVERSITY OF HONG KONG |
Hong Kong |
|
CN |
|
|
Family ID: |
52468071 |
Appl. No.: |
14/460732 |
Filed: |
August 15, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61866784 |
Aug 16, 2013 |
|
|
|
Current U.S.
Class: |
424/93.3 |
Current CPC
Class: |
A61P 1/16 20180101; A61K
35/747 20130101; Y02A 50/30 20180101; A61K 35/744 20130101; A61K
2035/115 20130101; Y02A 50/473 20180101; A61K 35/741 20130101; A61P
35/00 20180101; A61K 35/745 20130101; A61K 35/744 20130101; A61K
2300/00 20130101; A61K 35/745 20130101; A61K 2300/00 20130101; A61K
35/747 20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 35/741 20060101
A61K035/741 |
Claims
1. A probiotic composition comprising an effective amount of a
combination of bacteria, wherein the combination of bacteria
comprises (a) heat-inactivated Lactobacillus acidophilus (Moro)
Hansen and Mocqu (ATCC 53103), (b) viable Escherichia coli Nissle
1917, and (c) heat-inactivated bacteria selected from
Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium
infantis, Lactobacillus acidophilus, Lactobacillus plantarum,
Lactobacillus paracasei, Lactobacillus bulgaricus, Streptococcus
thermophiles, and any combinations thereof.
2. The probiotic composition of claim 1, wherein the combination of
bacteria is effective to reduce hepatocellular carcinoma (HCC)
growth compared to control growth.
3. The probiotic composition of claim 1, wherein the bacteria (a),
(b), and (c) are each independently present in the probiotic
composition at a concentration of about 20-55 weight percent.
4. A method of treating a subject, the method comprising orally
administering the probiotic composition of claim 1 to a subject at
risk of or diagnosed with hepatocellular carcinoma (HCC).
5. The method of claim 4, wherein the probiotic composition is
administered daily.
6. The method of claim 4, wherein the probiotic composition is
administered daily for at least one month.
7. The method of claim 4, wherein the subject is diagnosed with
HCC.
8. The method of claim 4, wherein the subject is at risk of
HCC.
9. The method of claim 8, wherein the subject is diagnosed with
hepatitis B virus, hepatitis C virus, or both.
10. The method of claim 8, wherein the subject has been exposed to
aflatoxin B1 (AFB1).
11. The method of claim 8, wherein the subject is diagnosed with
alcoholism.
12. The method of claim 4 further comprising, prior to
administering the probiotic composition, diagnosing the subject as
having HCC.
13. The method of claim 4 further comprising, prior to
administering the probiotic composition, diagnosing the subject as
at risk of HCC.
14. The method of claim 13, wherein diagnosing the subject as at
risk of HCC comprises diagnosing the subject as having hepatitis B
virus, hepatitis C virus, or both.
15. The method of claim 13, wherein diagnosing the subject as at
risk of HCC comprises diagnosing the subject as having been exposed
to aflatoxin B1 (AFB1).
16. The method of claim 13, wherein diagnosing the subject as at
risk of HCC comprises diagnosing the subject as having
alcoholism.
17. A method of treating a subject, the method comprising orally
administering a probiotic composition to a subject at risk of or
diagnosed with hepatocellular carcinoma (HCC), the probiotic
composition comprising an effective amount of a combination of
bacteria, wherein the combination of bacteria comprises (a)
heat-inactivated Lactobacillus acidophilus (Moro) Hansen and Mocqu
(ATCC 53103), (b) viable Escherichia coli Nissle 1917, and (c)
heat-inactivated bacteria selected from Bifidobacterium breve,
Bifidobacterium longum, Bifidobacterium infantis, Lactobacillus
acidophilus, Lactobacillus plantarum, Lactobacillus paracasei,
Lactobacillus bulgaricus, Streptococcus thermophiles, and any
combinations thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. provisional
application Ser. No. 61/866,784, filed Aug. 16, 2013, which is
herein incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] Hepatocellular carcinoma (HCC) is the fifth most prevalent
cancer and the third leading cause of all cancer-related deaths in
the world (Ferlay, J., et al., Estimates of worldwide burden of
cancer in 2008: GLOBOCAN 2008. International journal of cancer,
2010. 127(12): p. 2893-2917). More than 600,000 deaths are reported
globally each year. HCC affects all segments of the world
population (El-Serag, H. B. and K. L. Rudolph, Hepatocellular
carcinoma: epidemiology and molecular carcinogenesis.
Gastroenterology, 2007. 132(7): p. 2557-2576). Approximately 75% of
HCC patients are concentrated in Asia, but a recent trend of rising
rates of HCC has been reported in several developed countries
including Europe and the United States (Venook, A. P., et al., The
incidence and epidemiology of hepatocellular carcinoma: a global
and regional perspective. The Oncologist, 2010. 15 (Supplement 4):
p. 5-13).
[0003] The current standard of care can be classified into 3 broad
categories including (1) surgical treatment; (2) local ablation
therapy; and (3) chemotherapy. Surgical treatments include hepatic
resection and liver transplantation. Non-surgical treatments
include local ablation therapies, of which percutaneous ethanol
injection (PEI) and radiofrequency ablation (RFA) are most commonly
practiced; and chemotherapy which is either administered via
transarterial chemoembolization (TACE) or systemically. Standard
treatments involve high costs. For example, molecular targeted
therapy using sorafenib as recommended by NCCN/AASLD/JSH/ESMO
Guideline is expensive, with an average cost around US$6,000 per
month.
[0004] Oral consumption of viable probiotics is one of the many
alternative experimental cancer treatments that have been reported
in the literature. Probiotics are defined as live microorganisms
that, when administered in adequate amounts, confer a health
benefit on the host. Efficacy of probiotics on cancer development
is suggested by several lines of scientific evidence and
hypotheses, including the increase in immune cell activation,
increase of the antitumor surveillance immune activity, promotion
of anti-inflammation, and the suppression of bacteria converting
procarcinogens (de LeBlanc, A. d. M., C. Matar, and G. Perdigon,
The application of probiotics in cancer. British Journal of
Nutrition, 2007. 98(S1): p. 5105-5110). Most data were obtained
from cases of colon and breast carcinomas. In the former,
prevention was first achieved in animal models and was ascribed to
the significant anti-inflammatory effect of probiotics (Perdigon,
G., et al., Role of yoghurt in the prevention of colon cancer.
European journal of clinical nutrition, 2002. 56: p. S65). While in
the case of breast cancer, suggested beneficial effects of
probiotics appear to be mediated by an increase of the antitumor
surveillance immune activity (de Moreno de LeBlanc, A., et al.,
Effects of milk fermented by Lactobacillus helveticus R389 on
immune cells associated to mammary glands in normal and a breast
cancer model. Immunobiology, 2005. 210(5): p. 349-358).
[0005] In the context of liver cancer, there are reports regarding
the use of probiotic supplement (viable Lactobacillus rhamnosus
LC705 and Propionibacterium freudenreichii subsp. Shermani) as a
dietary approach to decrease the risk of liver cancer. The proposed
mechanism is related to blocking intestinal absorption of
carcinogen aflatoxin B1 by binding to the aflotoxin, and thereby
reducing the biologically effective dose of dietary aflatoxin
exposure (El-Nezami, H. S., et al., Probiotic supplementation
reduces a biomarker for increased risk of liver cancer in young men
from Southern China. The American journal of clinical nutrition,
2006. 83(5): p. 1199-1203).
[0006] There is substantial evidence that some probiotics can
provide benefits by modulating immune functions, including
modulating T helper cell response. Depending on the disease model,
these probiotics may induce T-cell hyporesponsiveness (Braat, H.,
et al., Lactobacillus rhamnosus induces peripheral
hyporesponsiveness in stimulated CD4+ T cells via modulation of
dendritic cell function. The American journal of clinical
nutrition, 2004. 80(6): p. 1618-1625), up-regulating or
down-regulating production of cytokines relating to Th1/Th2
response (Valio Ltd, R.D.a.S., LGG.RTM. Summatim, in Health effects
of LGG2009; Mutaflor. Most Recent Mutaflor Evidence and Clinical
Studies (2006-2010). 2010; Available on
webpagemutaflor.ca/health-care-professionals/mutaflor-clinical-studies/;
Sigma-Tau Pharmaceuticals, I. VSL#3.RTM. references. 2012;
Available on webpage vsl3.com/hcp/references.asp). Escherichia coli
Nissle 1917 (EcN) has been clinically studied for use in ulcerative
colitis, Crohn's Disease, chronic constipation, prolonged diarrhea,
irritable bowel syndrome and pouchitis (Mutaflor. Most Recent
Mutaflor Evidence and Clinical Studies (2006-2010). 2010; Available
on
webpagemutaflor.ca/health-care-professionals/mutaflor-clinical-studies/).
Lactobacillus acidophilus (Moro) Hansen and Mocquot strain GG (ATCC
53103) (LGG) has been shown to enhance antibody formation during
viral infection and respiratory infections, in addition to treating
the above gastrointestinal disorder (Valio Ltd, R.D.a.S., LGG.RTM.
Summatim, in Health effects of LGG2009). VSL#3.RTM. (Sigma-Tau) is
also widely used in clinical management of ulcerative colitis,
irritable bowel syndrome, and ileal pouch (Sigma-Tau
Pharmaceuticals, I. VSL#3.RTM. references. 2012; Available on
webpage vsl3.com/hcp/references.asp). In animal models, VSL#3.RTM.
has shown protective efficacy in delaying transition to dysplasia
in colitis-associated colorectal by attenuating various
inflammatory-associated parameters (Appleyard, C. B., et al.,
Pretreatment with the probiotic VSL#3 delays transition from
inflammation to dysplasia in a rat model of colitis-associated
cancer. American Journal of Physiology-Gastrointestinal and Liver
Physiology, 2011. 301(6): p. G1004-G1013).
[0007] Apart from viable probiotics, health benefits of
heat-inactivated probiotics supplementation have also been studied,
although the disease model is not cancer. Probiotic strains such as
L. acidophilus L-92, L. brevis SBC8803, L. casei Shirota, L.
gasseri OLL2809, L. paracasei KW3110, L. pentosus S-PT84 still show
immunomodulatory functions, including affecting IgE production, and
affecting Th1/Th2 cytokine production even after heat treatment in
animal models of sensitization and allergic manifestations
(Sashihara, T., N. Sueki, and S. Ikegami, An analysis of the
effectiveness of heat-killed lactic acid bacteria in alleviating
allergic diseases. Journal of dairy science, 2006. 89(8): p.
2846-2855). Meanwhile, non-probiotic bacteria have been
heat-attenuated and used in cancer studies in the form of cancer
vaccines. Examples of this include Coley's toxins, a mixture of
heat-killed S. pyogenes and S. marcescens, where attenuated
bacteria have been administered systemically to induce severe
inflammatory reaction against tumor by bacteria's endotoxin and
induce apoptosis. Though it is mainly used in the treatment of
sarcomas, lymphomas and melanomas (Cann, S. H., J. Van Netten, and
C. Van Netten, Dr William Coley and tumour regression: a place in
history or in the future. Postgraduate medical journal, 2003.
79(938): p. 672-680), its use in HCC patients has been reported in
one study as an adjuvant treatment. However, this kind of treatment
could be followed by severe fever with high risk of patient's death
(Richardson, M. A., et al., Coley toxins immunotherapy:a
retrospective review. Alternative therapies in health and medicine,
1999. 5(3): p. 42).
BRIEF SUMMARY OF THE INVENTION
[0008] It is an object of the present invention to provide
probiotic compositions that can treat cancer by, for example,
inhibiting the growth of the cancer. It is another object of the
invention to provide methods of treating cancer with probiotic
compositions.
[0009] It is desirable to prepare a probiotic formulation which
would act as a food supplement and therapeutic agent that assist in
reducing growth of hepatocellular carcinoma (HCC). To that end, the
disclosed probiotic formulations are useful as a food supplement
and therapeutic agent, and additionally are particularly useful for
reducing HCC growth.
[0010] Aspects of the present invention provide probiotic
compositions comprising an effective amount of a combination of
bacteria, where the combination of bacteria comprises (a)
heat-inactivated Lactobacillus acidophilus (Moro) Hansen and Mocqu
(ATCC 53103), (b) viable Escherichia coli Nissle 1917, and (c)
heat-inactivated VSL#3.RTM.. The combination of bacteria can be
effective to reduce hepatocellular carcinoma (HCC) growth compared
to control growth. The bacteria (a), (b), and (c) can each
independently be present in the probiotic composition at a
concentration of about 20-55 weight percent.
[0011] VSL#3.RTM. comprises Bifidobacterium breve, Bifidobacterium
longum, Bifidobacterium infantis, Lactobacillus acidophilus,
Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus
bulgaricus and Streptococcus thermophiles.
[0012] In at least one embodiment, the present invention provides
probiotic compositions comprising an effective amount of a
combination of bacteria, wherein the combination of bacteria
comprises (a) heat-inactivated Lactobacillus acidophilus (Moro)
Hansen and Mocqu (ATCC 53103), (b) viable Escherichia coli Nissle
1917, and (c) heat-inactivated bacteria selected from the group
consisting of Bifidobacterium breve, Bifidobacterium longum,
Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus
plantarum, Lactobacillus paracasei, Lactobacillus bulgaricus,
Streptococcus thermophiles and combinations thereof.
[0013] Aspects of the present invention also provide methods of
treating hepatocellular carcinoma (HCC). The methods comprise
orally administering a probiotic composition to a subject at risk
of or diagnosed with HCC. The probiotic composition can be
administered in any effective schedule. For example, and not meant
to be limiting, the probiotic composition can be administered
daily. The probiotic composition can be administered daily for at
least one month. The subject can be diagnosed with HCC. The subject
can be at risk of HCC. The subject can be a subject diagnosed with
hepatitis B virus, hepatitis C virus, or both. The subject can be a
subject exposed to aflatoxin B1 (AFB1). The subject can be a
subject diagnosed with alcoholism.
[0014] The methods can further comprise, prior to administering the
probiotic composition, diagnosing the subject as having HCC. The
methods can further comprise, prior to administering the probiotic
composition, diagnosing the subject as at risk of HCC. Diagnosing
the subject as at risk of HCC can comprise diagnosing the subject
as having hepatitis B virus, hepatitis C virus, or both. Diagnosing
the subject as at risk of HCC can comprise diagnosing the subject
as having been exposed to aflatoxin B1 (AFB1). Diagnosing the
subject as at risk of HCC can comprise diagnosing the subject as
having alcoholism.
[0015] Also disclosed are methods comprising diagnosing the subject
as having or being at risk of HCC and orally administering a
probiotic composition to the subject diagnosed as having or being
at risk of HCC.
[0016] The probiotic composition can be any of the disclosed
probiotic compositions. For example, and not meant to be limiting,
the probiotic composition can comprise an effective amount of a
combination of bacteria, where the combination of bacteria
comprises (a) heat-inactivated Lactobacillus acidophilus (Moro)
Hansen and Mocqu (ATCC 53103), (b) viable Escherichia coli Nissle
1917, and (c) heat-inactivated bacteria selected from the group
consisting of Bifidobacterium breve, Bifidobacterium longum,
Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus
plantarum, Lactobacillus paracasei, Lactobacillus bulgaricus,
Streptococcus thermophiles and combinations thereof. The
combination of bacteria can be effective to reduce hepatocellular
carcinoma (HCC) growth compared to control growth. The bacteria
(a), (b), and (c) can each independently present in the probiotic
composition at a concentration of about 20-55 weight percent.
[0017] In at least one embodiment, the present invention provides a
method of treating a subject comprising orally administering a
probiotic composition to a subject at risk of or diagnosed with
hepatocellular carcinoma (HCC), the probiotic composition
comprising an effective amount of a combination of bacteria,
wherein the combination of bacteria comprises (a) heat-inactivated
Lactobacillus acidophilus (Moro) Hansen and Mocqu (ATCC 53103), (b)
viable Escherichia coli Nissle 1917, and (c) heat-inactivated
bacteria selected from the group consisting of Bifidobacterium
breve, Bifidobacterium longum, Bifidobacterium infantis,
Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus
paracasei, Lactobacillus bulgaricus, Streptococcus thermophiles and
combinations thereof.
[0018] Additional advantages of the disclosed method and
compositions will be set forth in part in the description which
follows, and in part will be understood from the description, or
may be learned by practice of the disclosed method and
compositions. The advantages of the disclosed method and
compositions will be realized and attained by means of the elements
and combinations particularly pointed out in the appended claims.
It is to be understood that both the foregoing general description
and the following detailed description are exemplary and
explanatory only and are not restrictive of the invention as
claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate several
embodiments of the disclosed method and compositions and together
with the description, serve to explain the principles of the
disclosed method and compositions.
[0020] FIG. 1 is a graph of the ratio of tumor weight to body
weight after different treatments. The graph shows a smaller tumor
burden after consumption of probiotics as food supplement in a
prevention model. Male 5-6 week old C57BL6/N mice were fed with
probiotics daily starting 1 week before subcutaneous injection of
mouse hepatoma cell line Hepa1-6. The animals were sacrificed 38
days after tumor injection to determine tumor:body weight ratio.
Tumor burden of probiotics groups were significantly smaller than
the control as determined by Tukey's multiple comparison test, but
were indifferent between heat-inactivated and viable bacteria.
*p<0.05. **p<0.01. ***p<0.001. C: negative control; cis:
cisplatin (0.25 mg/ml every 3 days); vd: heat-inactivated
VSL#3.RTM. (1010 cfu/d); ld: heat-inactivated LGG (108 cfu/d); eh:
viable high dose EcN (108 cfu/d).
[0021] FIGS. 2A-2F show graphs of the relative mRNA expression of T
helper cell subset markers, IFNG (FIG. 2A), TBET (FIG. 2B), TGFB-1
(FIG. 2C), FoxP3 (FIG. 2D), IL-17A (FIG. 2E), and ROR.gamma.t (FIG.
2F), in tumor tissues after consumption of probiotics as food
supplement in a prevention model. FIGS. 2G-2L show graphs of the
relative mRNA expression of angiogenic markers, Angopiotiein 1
(FIG. 2G), Angopiotiein 2 (FIG. 2H), KDR (FIG. 2I), FLT (FIG. 2J),
Pecam-1 (FIG. 2K), and VE-cadherin (FIG. 2L), in tumor tissues
after consumption of probiotics as food supplement in a prevention
model. Expression levels in probiotic groups were compared to
control by Dunnett's test. Reduced expression of angiogenic markers
could generally be observed along with decreased Treg marker
(TGFB-1) and Th17 marker (ROR.gamma.t) expression, but not with Th1
marker (IFNG, TBET) expression. *p<0.05, **p<0.01,
****p<0.0001. c: control; cis: cisplatin; vd: heat-inactivated
VSL#3.RTM.; ld: heat-inactivated LGG (108 cfu/d); eh: viable high
dose EcN (108 cfu/d).
[0022] FIG. 3A shows a graph of tumor size and FIG. 3B shows a
graph of tumor weight in mice inoculated with 1.times.10.sup.7
Hepa1-6 tumor cells subcutaneously and fed with a ProHep diet daily
starting from either 1-week before (p: ProHep prevention) or at the
same day (pt: ProHep treatment) of tumor inoculation. Cisplatin was
given as positive control every 3 days i.p.
[0023] FIG. 4A shows confocal images of whole tumor sections with
TO-PRO-3 staining for dead cells. Scale bar, 200 .mu.m. FIGS. 4B-4D
show immunostaining for representative tumor sections for Ki67
(FIG. 4B), caspase 3 (FIG. 4C) and GLUT-1 (FIG. 4D) (blue) and CD31
(red) are shown. Scale bar, 50 .mu.m.
[0024] FIG. 5A shows microvessel density of representative tumor
sections. Scale bar, 50 .mu.m. FIG. 5B shows images of
three-dimensional models obtained by confocal Z stacks, after
superimposition of multiple confocal planes (section thickness, 25
.mu.m).
[0025] FIGS. 6A-6H show gene expression by real-time quantitative
PCR (RT-qPCR) in whole-tumor lysates of IL17 (FIG. 6A), ROR.gamma.t
(FIG. 6B), TGF-.beta. (FIG. 6C), Angopiotiein 2 (FIG. 6D), FLT
(FIG. 6E), KDR (FIG. 6F), TEK (FIG. 6G), and VE-cadherin (FIG. 6H).
Hprt were used as reference genes. FIG. 6I shows the correlation of
differential gene expression by hierarchical gene clustering
analysis.
[0026] FIG. 7A shows immunostaining of IL-17 in representative
tumor sections. FIG. 7B shows tumor sizes over time in C57BL/6N
mice inoculated with tumor and injected with anti-IL-17 antibody
(200 .mu.g/day) or control rat IgG. Tumor sizes were monitored for
1 month. Mice were under either a control or a ProHep diet starting
from 1 week in advance of tumor inoculation. FIG. 7C shows flow
cytometry analyses of % MDSC in tumor.
[0027] FIG. 8A shows confocal images of tumor sections with IL-17
staining (blue), co-stained (red) with CD3 (left panel), CD11b
(middle panel) and MPO (right panel). Scale bar, 50 .mu.m. FIG. 8B
shows flow cytometry analyses % Th subset in tumor. FIG. 8C shows
correlation analysis of % Th17, MVD and tumor size. FIG. 8D shows
phenotyping of Th17 in tumor. FIG. 8E shows Th17 frequency in
various organs by flow cytometry.
[0028] FIGS. 9A-9C show a ProHep diet reduces the capacity of naive
CD4+ T cells to differentiate into Th17. Naive CD4+CD62L+ T cells
were isolated from mice gut under a control or a ProHep diet and
differentiated into Th1, Th2, Th17 or Treg cells in the presence of
anti-CD3 and anti-CD28 for 72 hours. FIG. 9A shows RT-qPCR analysis
of Tbet, Gata3, Roryt and Foxp3 mRNA expression. Expression is
presented relative to Gapdh expression. FIG. 9B shows cytokine
secretion measured by ELISA in supernatants of CD4+ T cells
differentiated for 3 days. FIG. 9C shows flow cytometry analysis of
% Th1(CD4+IFN-.gamma.+), Th2 (CD4+IL-4+) and Th17 (CD4+IL-17+)
derived from naive CD4+ T cells under polarizing conditions
assessed for 5 day culture. Representative data from three
independent experiments are shown.
DETAILED DISCLOSURE OF THE INVENTION
[0029] The disclosed method and compositions may be understood more
readily by reference to the following detailed description of
particular embodiments and the Examples included therein and to the
Figures and their previous and following description.
[0030] Disclosed are materials, compositions and components that
can be used for, can be used in conjunction with, can be used in
preparation for, or are products of the disclosed method and
compositions. These and other materials are disclosed herein, and
it is understood that when combinations, subsets, interactions,
groups, etc. of these materials are disclosed that while specific
reference of each various individual and collective combinations
and permutation of these compounds may not be explicitly disclosed,
each is specifically contemplated and described herein. For
example, if a probiotic composition is disclosed and discussed and
a number of modifications that can be made to a number of
components including the probiotic bacteria are discussed, each and
every combination and permutation of probiotic bacteria and the
modifications that are possible are specifically contemplated
unless specifically indicated to the contrary. Thus, if a class of
bacteria A, B, and C are disclosed as well as a class of food D, E,
and F and an example of a combination components, A-D is disclosed,
then even if each is not individually recited, each is individually
and collectively contemplated. Thus, is this example, each of the
combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are
specifically contemplated and should be considered disclosed from
disclosure of A, B, and C; D, E, and F; and the example combination
A-D. Likewise, any subset or combination of these is also
specifically contemplated and disclosed. Thus, for example, the
sub-group of A-E, B-F, and C-E are specifically contemplated and
should be considered disclosed from disclosure of A, B, and C; D,
E, and F; and the example combination A-D. Further, each of the
materials, compositions, components, etc. contemplated and
disclosed as above can also be specifically and independently
included or excluded from any group, subgroup, list, set, etc. of
such materials. These concepts apply to all aspects of this
application including, but not limited to, steps in methods of
making and using the disclosed compositions. Thus, if there are a
variety of additional steps that can be performed it is understood
that each of these additional steps can be performed with any
specific embodiment or combination of embodiments of the disclosed
methods, and that each such combination is specifically
contemplated and should be considered disclosed.
[0031] A probiotic formulation, termed ProHep, useful as a food
supplement and therapeutic agent for reducing HCC tumor growth, is
contemplated. The probiotic formulation comprises a mixture of
beneficial probiotic microflora comprising (a) heat-inactivated
Lactobacillus acidophilus (Moro) Hansen and Mocqu (ATCC 53103), (b)
viable Escherichia coli Nissle 1917, and (c) heat-inactivated
VSL#3.RTM.. VSL#3.RTM. is a commercially available product traded
under the name VSL#3.RTM. and comprised of 8 different bacterial
strains: Bifidobacterium breve, Bifidobacterium longum,
Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus
plantarum, Lactobacillus paracasei, Lactobacillus bulgaricus and
Streptococcus thermophiles. It was determined that this probiotic
formulation can reduce HCC tumor growth.
[0032] The major shortcoming of current HCC treatment is the high
healthcare cost. For example, molecular targeted therapy using
sorafenib, as recommended by the NCCN/AASLD/JSH/ESMO Guideline, is
expensive, with an average cost around US$6,000 per month (Cabrera,
R. and D. Nelson, Review article: the management of hepatocellular
carcinoma. Alimentary pharmacology & therapeutics, 2010. 31(4):
p. 461-476), so it can only be performed in countries with
extensive financial resources for healthcare services (Benson 3rd,
A., et al., NCCN clinical practice guidelines in oncology:
hepatobiliary cancers. Journal of the National Comprehensive Cancer
Network: JNCCN, 2009. 7(4): p. 350; Jelic, S., Hepatocellular
carcinoma: ESMO clinical recommendations for diagnosis, treatment
and follow-up. Annals of Oncology, 2009. 20(suppl 4): p. iv41-iv45;
Izumi, N., Diagnostic and treatment algorithm of the Japanese
society of hepatology: a consensus-based practice guideline.
Oncology, 2010. 78(Suppl. 1): p. 78-86; Bruix, J. and M. Sherman,
Management of hepatocellular carcinoma: an update. Hepatology,
2011. 53(3): p. 1020-1022). Published cost analysis of HCC in the
United States indicates that the mean 5-year net cost is US$45,000
for an average HCC patient per month (Yabroff, K. R., et al., Cost
of care for elderly cancer patients in the United States. Journal
of the National Cancer Institute, 2008. 100(9): p. 630-641).
Meanwhile, probiotics are commonly found in dairy food products,
and the cost is only approximately US$86.00 per month for buying
patented commercial products such as VSL#3.RTM.. Thus, the
disclosed effective probiotics offer a cheaper approach in HCC
intervention.
[0033] Probiotic oral supplementation, whether in viable or
heat-inactivated form, was not previously used to treat HCC and was
not previously known to reduce HCC growth. Further, although
probiotics had shown some general immunomodulatory effects, T
helper cell related immunomodulatory effects in HCC tumor
microenvironment and its relationship to HCC prevention and
intervention were not studied prior to the present work.
Specifically, it was not previously known that LGG, EcN and
VSL#3.RTM. could have an effect on HCC.
[0034] The disclosed compositions and methods can also be used to
treat those at risk of developing HCC. Etiological factors of HCC
include chronic infections with the hepatitis B virus (HBV) or
hepatitis C virus (HCV), exposure to dietary aflatoxin B1 (AFB1),
and alcohol consumption. Among which, HBV is responsible for
50%-80% of HCC cases worldwide, whereas 10%-25% of cases are
thought to be a result of HCV infection (Venook, A. P., et al., The
incidence and epidemiology of hepatocellular carcinoma: a global
and regional perspective. The Oncologist, 2010. 15(Supplement 4):
p. 5-13; Block, T. M., et al., Molecular viral oncology of
hepatocellular carcinoma. Oncogene, 2003. 22(33): p. 5093-5107;
Anthony, P., Hepatocellular carcinoma: an overview. Histopathology,
2001. 39(2): p. 109-118). The disclosed probiotic compositions can
reduce the risk of developing HCC by modulating the immune system
by oral consumption of the probiotics before HCC is developed.
Thus, hepatitis carriers can be treated with the disclosed
probiotic compositions to help reduce their risk of developing HCC.
The World Health Organization (WHO) estimates that more than 520
million people in the world are chronic hepatitis virus carriers
who are at risk of developing liver cirrhosis and/or liver cancer
(WHO. Hepatitis B Global Alert and Response. 2013 [cited 2013;
Available on webpage
who.int/csr/disease/hepatitis/whocdscsrlyo20022/en/index3.html;
WHO. Hepatitis C. Global Alert and Response. 2013; Available on
webpage
who.int/csr/disease/hepatitis/whocdscsrlyo2003/en/index4.html).
Given the average cost for monthly probiotic consumption is around
US$68.30 (Ferring. VSL#3.RTM. ordering. 2013; Available on webpage
vsl3.co.uk/order_vsl3; Mutaflor. Buy Mutaflor. 2013; Available on
webpage mutaflor.ca/products-page/; verkkoapteekki, Gefilus. 2013),
the estimated market size for a treatment of 45 days with the
disclosed probiotics is about US$44 billion per year.
[0035] The disclosed compositions and methods allow the source of
chronic microbial challenge to the host to be targeted, which will
help to modulate T helper cell related immune response in tumor
microenvironment and reduce liver cancer progression. Disclosed are
methods for reducing HCC tumor growth using probiotics and methods
to reduce tumor growth using a new mixture of probiotics (ProHep).
By preventing or treating problems associated with tumor growth in
a patient, subsequent health problems can be prevented or
mitigated, which can result in reduced health care costs for the
patient in the future.
[0036] Some of the disclosed probiotic compositions are mixtures of
bacteria. For example, some probiotic compositions can be mixtures
of several commercially available or public domain bacteria,
namely, Lactobacillus acidophilus (Moro) Hansen and Mocquot strain
GG (ATCC 53103), Escherichia coli Nissle 1917 (commercially
available product traded under the name Mutaflor), VSL#3.RTM.
(commercially available product traded under the name VSL#3.RTM.).
The bacterial mixture is a mix of viable and heat-inactivated
bacteria, the mixture can be referred to as ProHep. The probiotic
components can also be formulated in two or more separate
compositions. Such separate compositions can be administered
together or separately, at the same time or at different times. The
disclosed compositions can further comprise, or can be administered
with, other components of compositions. For example, the disclosed
probiotic compositions can be combined with food or can be
administered with food.
[0037] While not wishing to be limited to a particular manner of
operation, the disclosed compositions and methods may reduce HCC
tumor growth by influencing T helper cells--related immune
responses in the tumor microenvironment and angiogenesis (Sung C Y,
L. N., El-Nezami H., Regulation of T helper 17 by bacteria: an
approach for the treatment of hepatocellular carcinoma.
International journal of Hepatology, 2012. 2012(Article ID 439024):
p. 8).
[0038] It is to be understood that the disclosed method and
compositions are not limited to specific synthetic methods,
specific analytical techniques, or to particular reagents unless
otherwise specified, and, as such, may vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only and is not intended to be
limiting.
Probiotic Compositions
[0039] Disclosed are probiotic compositions useful for treating
hepatocellular carcinoma (HCC). The disclosed probiotic
compositions are probiotic formulations useful as a food supplement
and therapeutic agent for reducing HCC risk and/or growth and
generally comprise a mixture of beneficial probiotic microflora. It
was observed that certain combinations of probiotic bacteria can
reduce the risk and/or effect of HCC. Some of the probiotic
compositions comprise heat-inactivated Lactobacillus acidophilus
(Moro) Hansen and Mocqu (ATCC 53103), viable Escherichia coli
Nissle 1917, and heat-inactivated VSL#3.RTM.. For example, the
probiotic composition can comprise Lactobacillus acidophilus (Moro)
Hansen and Mocquot strain GG (ATCC 53103) (heat-inactivated,
2.times.1010 cfu/d); Bifidobacterium breve, Bifidobacterium longum,
Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus
plantarum, Lactobacillus paracasei, Lactobacillus bulgaricus and
Streptococcus thermophiles, commercially known as VSL#3.RTM.
(heat-inactivated, total dose 1.8.times.1012 cfu/d); and
Escherichia coli Nissle 1917 (viable, 5.times.1012 cfu/d). The
combination of bacteria can be effective to reduce hepatocellular
carcinoma (HCC) growth compared to control growth. The combination
of bacteria can be effective to reduce hepatocellular carcinoma
(HCC) risk compared to control risk. The bacteria (a), (b), and (c)
can each be independently present in the probiotic composition at a
concentration of about 20-55 weight percent.
[0040] Lactobacillus acidophilus (Moro) Hansen and Mocqu (ATCC
53103) are bacteria that are known to modulate production of
cytokines relating to Th1/Th2 response, induce T-cell
hyporesponsiveness and reduce inflammation in systemic and gut
diseases models.
[0041] Escherichia coli Nissle 1917 are known bacteria that can
induce T-cell hyporesponsiveness and promote anti-inflammation.
[0042] VSL#3.RTM. (Sigma-Tau) are a patented, commercially
available probiotic mixture composed of eight bacteria:
Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium
infantis, Lactobacillus acidophilus, Lactobacillus plantarum,
Lactobacillus paracasei, Lactobacillus bulgaricus, and
Streptococcus thermophiles. VSL#3.RTM. may promote
anti-inflammation by increasing production of anti-inflammatory
cytokines, expand Treg pool and decrease levels of proinflammatory
cytokines.
[0043] These three probiotic strains and probiotic mixture may
each, individually, be present in the probiotic composition at a
concentration from about 20 to about 55 weight percent. Examples of
combinations of concentrations of Lactobacillus acidophilus (Moro)
Hansen and Mocqu (LGG), Escherichia coli Nissle 1917 (EcN), and
VSL#3.RTM. in the probiotic composition are listed in Table 1.
TABLE-US-00001 TABLE 1 Concentrations of Probiotic Bacteria (in
weight percent of composition) Example No. LGG EcN VSL#3 .RTM. 1 20
20 20 2 20 20 25 3 20 20 30 4 20 20 35 5 20 20 40 6 20 20 45 7 20
20 50 8 20 20 55 9 20 25 20 10 20 30 20 11 20 35 20 12 20 40 20 13
20 45 20 14 20 50 20 15 20 55 20 16 25 20 20 17 30 20 20 18 35 20
20 19 40 20 20 20 45 20 20 21 50 20 20 22 55 20 20 23 30 20 25 24
30 20 30 25 30 20 35 26 30 20 40 27 30 20 45 28 30 20 50 29 30 25
20 30 30 30 20 31 30 35 20 32 30 40 20 33 30 45 20 34 30 50 20 35
40 20 25 36 40 20 30 37 40 20 35 38 40 20 40 39 40 25 20 40 40 30
20 41 40 35 20 42 40 40 20 43 50 20 25 44 50 20 30 45 50 25 20 46
50 30 20 47 55 20 25 48 55 25 20
[0044] The ingredients of the probiotic composition can be mixed
together by conventional methods and formed into, for example,
tablets for oral administration. Alternatively, the ingredients can
be mixed together and placed into gelatin capsules. The disclosed
probiotic formulations can also contain conventional food
supplement fillers and extenders such as, for example, rice flour.
Conveniently, the probiotic formulation can be taken orally at a
dosage rate ranging from about 5 milligrams per day to about 50
milligrams per day.
[0045] The disclosed probiotic compositions can be formulated as
edible, cosmetic or pharmaceutical products. The probiotic
compositions can comprise, in addition to the probiotic bacteria,
one or more other active agents and/or cosmetically acceptable
excipients (in the case of a cosmetic composition),
pharmaceutically acceptable excipients (in the case of a
pharmaceutical composition) or adequate edible ingredients (in the
case of an edible composition). Some probiotic compositions can
further comprise one or more active agents. For example, the
additional active agent or agents can be other probiotic bacteria
that are not antagonistic to the bacteria forming the disclosed
probiotic compositions. Depending on the formulation, the bacteria
be added as purified bacteria, as a bacterial culture, as part of a
bacterial culture, as a bacterial culture which has been
post-treated, and alone or together with suitable carriers or
ingredients. Prebiotics could also be added.
[0046] The disclosed probiotic compositions can be also included in
a variety of edible products, such as milk products, yogurt, curd,
cheese (e.g., quark, cream, processed, soft and hard), fermented
milk, milk powder, milk based fermented product, ice-cream, a
fermented cereal based product, milk based powder, a beverage, a
dressing, and a pet food. The term "edible product" is used herein
in its broadest meaning, including any type of product, in any form
of presentation, which can be ingested by an animal, but excluding
cosmetic, pharmaceutical, and veterinary products. Examples of
other edible products are meat products (e.g., liver paste,
frankfurter and salami sausages or meat spreads), chocolate
spreads, fillings (e.g., truffle, cream) and frostings, chocolate,
confectionery (e.g., caramel, candy, fondants or toffee), baked
goods (cakes, pastries), sauces and soups, fruit juices and coffee
whiteners. The compositions of the invention could be also used as
an ingredient in other food products. Particularly useful edible
products are functional foods.
[0047] Accordingly, also disclosed are edible compositions that
contain the disclosed probiotic composition together with other
edible ingredients. The term "edible ingredients" refers to
ingredients which are fit to be eaten, i.e. to be used as food, by
an animal, preferably but not limited to humans, cattle or pet
animals.
[0048] Often, probiotic compositions such as those disclosed herein
are considered as dietary supplements. Dietary supplements, also
known as food supplements or nutritional supplements, provide
beneficial ingredients that are not usually ingested in the normal
diet. Mostly, dietary supplements are considered as food products,
but sometimes they are defined as drugs, natural health products,
or nutraceutical products. As used herein, dietary supplements also
include nutraceuticals. Dietary supplements are usually sold "over
the counter", i.e., without prescription. Some forms of the
disclosed probiotic compositions are dietary supplements.
[0049] If the probiotic composition is a dietary supplement, it can
be administered as such, can be mixed with a suitable drinkable
liquid, such as water, yoghurt, milk or fruit juice, or can be
mixed with solid or liquid food. In this context the dietary
supplement can be in the form of tablets, pills, capsules,
lozenges, granules, powders, suspensions, sachets, pastilles,
sweets, bars, syrups and corresponding administration forms,
usually in the form of a unit dose. For example, the dietary
supplement comprising the probiotic composition can be administered
in the form of tablets, lozenges, capsules or powders, manufactured
in conventional processes of preparing dietary supplements.
[0050] The bacteria forming the disclosed probiotic compositions
can be in the form of viable cells or non-viable cells. In some
embodiments, Lactobacillus acidophilus (Moro) Hansen and Mocqu and
VSL#3.RTM. are non-viable and Escherichia coli Nissle 1917 are
viable cells. Non-viable cells can be produced by, for example,
thermally killed micro-organisms or micro-organisms killed by
exposure to altered pH, sonication, radiation or subjection to
pressure. With non-viable cells, product preparation is simpler,
cells may be incorporated easily into commercial products and
storage requirements are much less limited than viable cells.
[0051] The amount or concentration of bacteria used in the
probiotic composition generally can be noted as the amount of
colony forming units (cfu) for each strain or combination of
strains in the composition. Generally, an effective amount of the
bacteria will be an amount that is high enough to significantly
modify the condition to be treated in a positive way but low enough
to avoid serious side effects (at a reasonable benefit/risk ratio),
within the scope of sound medical judgment. An effective amount of
the probiotic bacteria can be determined by the skilled artisan and
will vary with the particular goal to be achieved, the age and
physical condition of the patient being treated, the severity of
the underlying disorder, and the final formulation. For example,
the bacteria can be present in an amount from about 10.sup.5 cfu/g
to about 10.sup.12 cfu/g, preferably in an amount from about
10.sup.7 cfu/g to about 10.sup.11 cfu/g. The term "colony forming
unit" ("cfu") refers to the number of bacterial cells as revealed
by microbiological counts on agar plates. In some probiotic
compositions, the probiotic composition comprises between
10.sup.7-10.sup.10 cfu/g.
[0052] The bacteria for use in the disclosed probiotic compositions
can be produced by, for example, cultivating the bacteria in a
suitable medium and under suitable conditions. The bacteria can be
cultivated alone to form a pure culture, or as a mixed culture
together with other microorganisms, or by cultivating bacteria of
different types separately and then combining them in the desired
proportions. After cultivation, the cell suspension can be
recovered and used as such or treated in the desired manner, for
instance, by concentrating or freeze-drying, to be further employed
in the preparation of the products. Sometimes the probiotic
preparation can be subjected to an immobilization or encapsulation
process in order to improve the shelf life. Several techniques for
immobilization or encapsulation of bacteria are known in the
art.
[0053] Lactobacillus acidophilus (Moro) Hansen and Mocqu (ATCC
53103), Escherichia coli Nissle 1917, and VSL#3.RTM. can be
effective not only when combined in a single composition, but also
when used on their own, or in two or more different compositions
administered simultaneously, sequentially, or separately after a
certain period of time.
[0054] "Probiotics" are live microorganisms that when administered
in adequate amounts confer a beneficial health effect on the host.
Natural intestinal microflora include numerous probiotic
microorganisms. Heat-inactivated probiotics refer to probiotics
that have been killed by heating. The term "parts by weight," of a
particular element or component in a composition or article,
denotes the weight relationship between the element or component
and any other elements or components in the composition or article
for which a part by weight is expressed. Thus, in a compound
containing 2 parts by weight of component X and 5 parts by weight
component Y, X and Y are present at a weight ratio of 2:5, and are
present in such ratio regardless of whether additional components
are contained in the compound.
[0055] As used herein, a "weight percent" of a component, unless
specifically stated to the contrary, is based on the total weight
of the formulation or composition in which the component is
included. Thus, for example, a component at a concentration of
20-55 weight percent means that the component constitutes 20-55
percent of the total weight of the composition in which the
component is included.
Kits
[0056] The materials described above, as well as other materials,
can be packaged together in any suitable combination as a kit
useful for performing, or aiding in the performance of, the
disclosed methods. It is useful if the kit components in a given
kit are designed and adapted for use together in the disclosed
methods. For example, disclosed are kits for treating a subject at
risk of or diagnosed with HCC, the kits comprising heat-inactivated
Lactobacillus acidophilus (Moro) Hansen and Mocqu (ATCC 53103),
viable Escherichia coli Nissle 1917, and heat-inactivated
VSL#3.RTM..
Methods
[0057] Disclosed are methods of treating hepatocellular carcinoma
(HCC). The methods comprise orally administering a probiotic
composition to a subject at risk of or diagnosed with HCC. The
probiotic composition can be administered in any effective
schedule. For example, the probiotic composition can be
administered daily. The probiotic composition can be administered
daily for at least one month. The subject can be diagnosed with
HCC. The subject can be at risk of HCC. The subject can be a
subject diagnosed with hepatitis B virus, hepatitis C virus, or
both. The subject can be a subject exposed to aflatoxin B1 (AFB1).
The subject can be a subject diagnosed with alcoholism.
[0058] The methods can further comprise, prior to administering the
probiotic composition, diagnosing the subject as having HCC. The
methods can further comprise, prior to administering the probiotic
composition, diagnosing the subject as at risk of HCC. Diagnosing
the subject as at risk of HCC can comprise diagnosing the subject
as having hepatitis B virus, hepatitis C virus, or both. Diagnosing
the subject as at risk of HCC can comprise diagnosing the subject
as having been exposed to aflatoxin B1 (AFB1). Diagnosing the
subject as at risk of HCC can comprise diagnosing the subject as
having alcoholism.
[0059] Also disclosed are methods comprising diagnosing the subject
as having or being at risk of HCC and orally administering a
probiotic composition to the subject diagnosed as having or being
at risk of HCC.
[0060] Also disclosed are mixtures formed by performing or
preparing to perform the disclosed methods. For example, disclosed
are mixtures comprising heat-inactivated Lactobacillus acidophilus
(Moro) Hansen and Mocqu (ATCC 53103), viable Escherichia coli
Nissle 1917, heat-inactivated VSL#3.RTM.. The disclosed probiotic
formulations can also be mixtures with, for example, food, food
components, and conventional food supplement fillers and extenders
such as, for example, rice flour.
[0061] Whenever the methods involve mixing or bringing into contact
compositions or components or reagents, performing the methods
create a number of different mixtures. For example, if the method
includes 3 mixing steps, after each one of these steps a unique
mixture is formed if the steps are performed separately. In
addition, a mixture is formed at the completion of all of the steps
regardless of how the steps were performed. The present disclosure
contemplates these mixtures, obtained by the performance of the
disclosed methods as well as mixtures containing any disclosed
reagent, composition, or component, for example, disclosed
herein.
[0062] The terms "one day's treatment", "daily treatment", "daily
administration" and "daily intake" are used interchangeably and
mean the total dose of the probiotic composition to be taken by or
administered to the patient each day in the treatment period. If
the treatment is administered by the patient him/herself, the right
dose is preferably provided in a suitable container containing the
correct dose of the medical product. The total daily dose may
advantageously be divided into two or more containers containing
the right doses to be taken or administered during the day, for
example as the first and the last meal of the day. Alternatively,
the patient or the medical staff may measure the right dose for
each administration from a larger container comprising the
probiotic composition.
[0063] The term "reduce the risk" of a condition or event refers to
either or both (a) reducing the likelihood or probability of the
condition or event occurring or coming into existence compared to a
standard or reference likelihood or probability of the condition or
event occurring or coming into existence and (b) reducing or
ameliorating the amount, size, concentration, severity, rate,
stage, progress, and/or other measure of the condition or event if
it occurs compared to a standard or reference amount, size,
concentration, severity, rate, stage, progress, and/or other
measure of the condition or event. For example, reducing the risk
of HCC refers to either or both (a) reducing the likelihood or
probability of HCC occurring and (b) reducing or ameliorating the
amount, size, severity, rate of growth, stage, progress, and/or
other measure of HCC severity or stage if HCC occurs. Any one or
combination of the effects related to risk can be considered a
reduction in the risk of the condition or event. For example, if
the rate of growth of HCC once it occurs is lower than a standard
or reference rate of growth (such as the rate of growth of an
untreated subject with HCC) then the risk of HCC can be said to
have been reduced. As another example, if treated subjects without
HCC develop HCC at a lower rate than a standard or reference rate
then the risk of HCC can be said to have been reduced.
[0064] The term "complete oral nutritional supplement" refers to
products and formulations that can provide a subject with nutrition
orally that is nutritionally complete; that is, that can be used as
the sole source of nutrition.
[0065] The terms "high," "higher," "increases," "elevates," or
"elevation" refer to increases above basal or reference levels,
e.g., as compared to a control. The terms "low," "lower,"
"reduces," or "reduction" refer to decreases below basal or
reference levels, e.g., as compared to a control.
[0066] The term "reduce" means to lower or decrease in activity,
amount, size, weight, rate, stage, severity, expression, or the
like. This can be a complete reduction of activity, amount, size,
weight, rate, stage, severity, expression, or the like, or a
partial reduction of activity, amount, size, weight, rate, stage,
severity, expression, or the like. Reduction can be compared to a
control or to a standard level. Reduction can be 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,
75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100%.
[0067] The term "providing" as used herein refers to any means of
adding a compound, molecule, component, or composition to something
else, as well as to the act of such addition. Examples of providing
can include the use of pipettes, pipettemen, syringes, needles,
tubing, guns, etc. This can be manual or automated. It can include
transfection by any mean or any other means of providing nucleic
acids to dishes, cells, tissue, cell-free systems and can be in
vitro or in vivo.
[0068] The term "preventing" as used herein refers to administering
a composition prior to the onset of clinical symptoms of a disease
or condition so as to prevent a physical manifestation of
aberrations associated with the disease or condition.
[0069] The term "in need of treatment" as used herein refers to a
judgment made by a caregiver (e.g. physician, nurse, nurse
practitioner, or individual in the case of humans; veterinarian in
the case of animals, including non-human mammals) that a subject
requires or will benefit from treatment. This judgment is made
based on a variety of factors that are in the realm of a care
giver's expertise, but that include the knowledge that the subject
is ill, or will be ill, as the result of a condition that is
treatable by the compounds of the invention.
[0070] As used herein, "subject" includes, but is not limited to,
animals, plants, bacteria, viruses, parasites and any other
organism or entity. The subject can be a vertebrate, more
specifically a mammal (e.g., a human, horse, pig, rabbit, dog,
sheep, goat, non-human primate, cow, cat, guinea pig or rodent), a
fish, a bird or a reptile or an amphibian. The subject can be an
invertebrate, more specifically an arthropod (e.g., insects and
crustaceans). The term does not denote a particular age or sex.
Thus, adult and newborn subjects, as well as fetuses, whether male
or female, are intended to be covered. A patient refers to a
subject afflicted with a disease or disorder. The term "patient"
includes human and veterinary subjects.
[0071] By "treatment" and "treating" is meant the medical
management of a subject with the intent to cure, ameliorate,
stabilize, or prevent a disease, pathological condition, or
disorder. This term includes active treatment, that is, treatment
directed specifically toward the improvement of a disease,
pathological condition, or disorder, and also includes causal
treatment, that is, treatment directed toward removal of the cause
of the associated disease, pathological condition, or disorder. In
addition, this term includes palliative treatment, that is,
treatment designed for the relief of symptoms rather than the
curing of the disease, pathological condition, or disorder;
preventative treatment, that is, treatment directed to minimizing
or partially or completely inhibiting the development of the
associated disease, pathological condition, or disorder; and
supportive treatment, that is, treatment employed to supplement
another specific therapy directed toward the improvement of the
associated disease, pathological condition, or disorder. It is
understood that treatment, while intended to cure, ameliorate,
stabilize, or prevent a disease, pathological condition, or
disorder, need not actually result in the cure, ameliorization,
stabilization or prevention. The effects of treatment can be
measured or assessed as described herein and as known in the art as
is suitable for the disease, pathological condition, or disorder
involved. Such measurements and assessments can be made in
qualitative and/or quantitative terms. Thus, for example,
characteristics or features of a disease, pathological condition,
or disorder and/or symptoms of a disease, pathological condition,
or disorder can be reduced to any effect or to any amount.
[0072] A cell can be in vitro. Alternatively, a cell can be in vivo
and can be found in a subject. A "cell" can be a cell from any
organism including, but not limited to, a bacterium.
[0073] The term "effective amount" of a composition refers to a
nontoxic but sufficient amount of the composition to provide the
desired result. As discussed elsewhere herein, the exact amount
required will vary from subject to subject, depending on the
species, age, and general condition of the subject, the mode of
administration, and the like. Thus, it is not possible to specify
an exact "effective amount." However, an appropriate effective
amount can be determined by one of ordinary skill in the art using
only routine experimentation.
[0074] The dosages or amounts of the disclosed probiotic
compositions are large enough to produce the desired effect in the
method by which delivery occurs. The dosage should not be so large
as to cause adverse side effects, such as unwanted cross-reactions,
anaphylactic reactions, and the like. Generally, the dosage will
vary with the age, condition, sex and extent of the disease in the
subject and can be determined by one of skill in the art. The
dosage can be adjusted by the individual physician based on the
clinical condition of the subject involved. The dose, schedule of
doses and route of administration can be varied.
[0075] By "pharmaceutically acceptable" is meant a material that is
not biologically or otherwise undesirable, i.e., the material can
be administered to a subject along with the selected compound
without causing any undesirable biological effects or interacting
in a deleterious manner with any of the other components of the
pharmaceutical composition in which it is contained.
[0076] All patents, patent applications, provisional applications,
and publications referred to or cited herein are incorporated by
reference in their entirety, including all figures and tables, to
the extent they are not inconsistent with the explicit teachings of
this specification.
[0077] Following are examples that illustrate procedures for
practicing the invention. These examples should not be construed as
limiting. All percentages are by weight and all solvent mixture
proportions are by volume unless otherwise noted.
Example 1
ProHep Treatment Reduces Tumor Size
[0078] The effect of a probiotic mixture of bacteria was tested on
an animal model of HCC. Male C57BL/6N mice (5-6 weeks-old) were fed
daily with a therapeutically effective amount of probiotics for a
total of 45 days, starting one week before subcutaneous tumor
inoculation with 1.times.10.sup.7 Hepa106 cells. Tumor size was
found to be significantly smaller in animals fed with both
heat-inactivated bacteria and viable bacteria (FIG. 1). Analysis on
VSL#3.RTM. groups revealed that smaller tumor size appears to be
correlated with lower expression level of Treg marker TGFB (R=0.5,
p<0.05), Th17 marker RORC (R=0.5, p<0.05), and angiogenic
markers ANGPT2 (R=0.6, p<0.05), TIE1 (R=0.6, p<0.01),
VE-cadherin (R=0.7, p<0.005), suggesting that probiotics
consumption may modulate T cell subsets in tumor and may this may
have an impact on tumor angiogenesis (FIGS. 2A-2L).
[0079] In another study, C57BL6/N mice were inoculated with
1.times.10.sup.7 Hepa1-6 tumor cells subcutaneously and tumor sizes
were monitored for 38 days with caliper. Mice were fed with a
ProHep diet daily starting from either 1-week before (p: ProHep
prevention) or at the same day (pt: ProHep treatment) of tumor
inoculation. Cisplatin, a common chemotherapeutic drug, was given
as positive control at a dose of 0.25 mg/mL every 3 days i.p. Tumor
volume was significantly smaller in probiotic groups after 1 month
(FIG. 3A). Tumors that were excised at the end of the experiment
had a smaller weight in prevention group as compared to control
(*p<0.05) (FIG. 3B). Statistical analyses by one-way ANOVA,
post-hoc Tukey's multiple comparison test.
Example 2
Tumor Growth is Limited by Increased Hypoxia
[0080] Confocal images of whole tumor sections with TO-PRO-3
staining for dead cells is shown in FIG. 4A. Scale bar, 200 .mu.m.
FIGS. 4B-4D show immunostaining for representative tumor sections
for Ki67 (FIG. 4B), caspase 3(FIG. 4C) and GLUT-1 (FIG. 4D) (blue)
and CD31 (red). Scale bar, 50 .mu.m. Significant increase in area
of cell death was observed in ProHep groups, however, there was no
significant difference in the number of proliferating cells (Ki67+)
and apoptotic cells (caspase-3+), suggesting that reduced tumor
size is not related to reduced growth and increased apoptosis, but
is related to increased necrosis. Meanwhile, significant increase
in hypoxic area (GLUT-1) was found in the ProHep prevention (p)
group, suggesting that reduced tumor size is likely to be related
to hypoxia-induced cell death.
Example 3
ProHep Inhibits Angiogenesis in a Subcutaneous HCC Model
[0081] Microvessel density of representative tumor sections is
shown in FIG. 5A. FIG. 5B shows images of three-dimensional models
obtained by confocal Z stacks, after superimposition of multiple
confocal planes. Significantly smaller microvessel density,
relative vascular area and vessel sprouting were observed in ProHep
groups as compared to control. (*p<0.05; **, p<0.01; ***,
p<0.001).
Example 4
ProHep Treatment Downregulates the Expression of Proangiogenic
Genes in Tumors
[0082] FIGS. 6A-6H show gene expression by real-time quantitative
PCR (RT-qPCR) in whole-tumor lysates. Hprt were used as reference
genes. *p<0.05; **, p<0.01; ***, p<0.001. FIG. 6I shows
the correlation of differential gene expression by hierarchical
gene clustering analysis. The smaller tumor burden in the ProHep
groups may be associated with lower expression level of Th17 (RORC,
IL-17) and angiogenic markers (VE-cadherin, ANG2, KDR, FLT, TEK and
TGFB).
Example 5
ProHep Prevents Tumor Outgrowth in an IL-17-Dependent Manner
[0083] FIG. 7A shows immunostaining of IL-17 in representative
tumor sections. Densities of IL-17+ cells were significantly
smaller in ProHep groups. C57BL/6N mice were inoculated with tumor
and injected with anti-IL-17 antibody (200 .mu.g/day) or control
rat IgG. Tumor sizes were monitored for 1 month. Mice were under
either a control or a ProHep diet starting from 1 week in advance
of tumor inoculation. As shown in FIG. 7B, ProHep failed to exhibit
any effect on tumor progression after IL-17 neutralization. C) Flow
cytometry analyses of % MDSC in tumor showed reduced infiltration
of MDSC in ProHep and IL-17 neutralization (FIG. 7C).
Example 6
ProHep Prevents Tumor Outgrowth is Associated with Th17
[0084] FIG. 8A shows confocal images of tumor sections with IL-17
staining (blue), co-stained (red) with CD3 (left panel), CD11b
(middle panel) and MPO (right panel). Scale bar, 50 .mu.m. FIG. 8B
shows flow cytometry analyses % Th subset in tumor. FIG. 8C shows
the correlation analysis of % Th17, MVD and tumor size. FIG. 8D
shows phenotyping of Th17 in tumor, and FIG. 8E shows the Th17
frequency in various organs by flow cytometry.
[0085] Immunostaining showed that the majority of IL-17+ cells in
tumors were found to be CD3+ cells. Since Th cells were reported to
be the major producer of IL-17, Th subset phenotyping was performed
by flow cytometry and found that Th17 subset was significantly
reduced in the tumor microenvironment, and their population were
found to be correlated with reduced MVD and tumor size. Th17 in
tumor has expressed migratory phenotype CCR6, suggesting these
cells may be recruited from periphery site. Flow cytometry analyses
showed a trend of reduced recruitment of Th17 from intestine to
tumor via circulation.
Example 7
A ProHep Diet Reduces the Capacity of Naive CD4+ T Cells to
Differentiate into Th17
[0086] Naive CD4+CD62L+ T cells were isolated from mouse gut under
a control or a ProHep diet and differentiated into Th1, Th2, Th17
or Treg cells in the presence of anti-CD3 and anti-CD28 for 72
hours. FIG. 9A shows RT-qPCR analyses of Tbet, Gata3, Roryt and
Foxp3 mRNA expression. Expression is presented relative to Gapdh
expression. Significant downregulation of Th17 transcription factor
Roryt was observed. FIG. 9B shows cytokine secretion measured by
ELISA in supernatants of CD4+ T cells differentiated for 3 days.
Production of IL-17 (right panel) was decreased while production of
IFN-.gamma. (left panel) and IL-4 (middle panel) from Th1 and Th2
was unaffected. FIG. 9C shows flow cytometry analyses of %
Th1(CD4+IFN-.gamma.+), Th2 (CD4+IL-4+) and Th17 (CD4+IL-17+)
derived from naive CD4+ T cells under polarizing conditions
assessed for a 5 day culture. Representative data from three
independent experiments are shown. Significant reduction of Th17
polarization was observed.
DISCUSSION
[0087] In this study, the use of probiotics in HCC treatment via
immunomodulation in the tumor microenvironment is described.
Probiotics are defined as live microorganisms that, when
administered in adequate amounts, confer a health benefit on the
host. Beneficial probiotic formulation (ProHep) comprising of (a)
heat-inactivated Lactobacillus acidophilus (Moro) Hansen and Mocqu
(ATCC 53103), (b) viable Escherichia coli Nissle 1917, and (c)
heat-inactivated VSL#3.RTM. was found to be effective to reduce
subcutaneous HCC growth in mice when administered orally on daily
basis for 38 days. Tumor size in the probiotic group was
significantly smaller than control group starting from day 31,
while the beneficial effect could be observed even earlier (day 28)
when ProHep was administered 1 week before tumor inoculation. Yet,
the anti-cancer effects of probiotic groups were less potent than
drug (cisplatin) treatment group.
[0088] The smaller tumor size in the probiotic group was unlikely
to be due to reduce cell proliferation but may be related to
increase in necrotic cell death, as immunohistochemistry staining
revealed that there were no significant differences in the number
of Ki67+ cells in tumor tissue, while there was larger area of cell
death (TO-PRO3) and no significant change in number of caspase 3+
apoptotic cells. Meanwhile, an increase in hypoxic (GLTU-1+) area
was observed in the probiotic group, suggesting that increase in
cell death was possibly related to increase hypoxia due to
reduction in angiogenesis. Indeed, mean microvessel density,
percentage area of blood vessel per tumor section and number of
vessel sprouts, as quantified PECAM-1+ cells, were all
significantly lower in tumors established in probiotic-treated mice
as compared to control group. RT-qPCR analysis of tumor in the
probiotic group has shown reduced expression of several angiogenic
genes, including ANG2, FLT-1, KDR, TIE, TGFB, where their
expression have been associated with reduced IL-17 and RORC
expression by hierarchical gene clustering, suggesting that Th17 or
IL-17 producing cells may be involved in the probiotics-mediated
effect on HCC growth. This notion was further supported by the
observation of reduced IL-17+ cells in tumor sections of the
probiotic group, and that similar reduction of tumor growth could
be observed in the probiotic group and control group after IL-17
antibody neutralization. Although several immune cells have been
reported by these findings, most IL-17+ cells were co-stained with
CD3, and the tumor infiltrating Th17 population has decreased in
the probiotic group. Flow cytometic analysis that show reduced
infiltration of angiogenic cells, such as myeloid derived
suppressor cells, into the tumor microenvironment after IL-17
neutralization and probiotic treatment. Together these data suggest
a role for probiotics in impairing tumor angiogenesis by modulating
immune cell infiltration via Th17.
[0089] Since a majority of Th17 cells in the tumor microenvironment
have an expressed migratory phenotype (CCR6+), it is possible that
Th17 was recruited systemically into the tumor microenvironment.
The gut seems to be the potential source of Th17, as a reduced Th17
population was found in the gut of the probiotic group by flow
cytometry, but not in other organs such as spleen, mesenteric lymph
node, blood, liver. Probiotics may reduce the ability of naive T
cells to differentiate into Th17 cells in the gut. Following
activation of naive T cells isolated from the gut of mice fed with
probiotics, there was reduced gene expression of Rorc, the
transcription factor of Th17, but not the transcription factor of
TH1, TH2 and Treg. In addition, there was reduced IL-17A production
from Th17 cells by ELISA, while IFN-.gamma. or IL-4 secretion from
TH1 and TH2 cells derived from mice under a probiotic diet were not
affected. Intracellular staining analysis by flow cytometry also
confirmed dampened IL-17A secretion from TH17 cells. Taken
together, these results suggest that probiotics selectively affect
TH17 cell differentiation in the gut and its recruitment into the
tumor microenvironment.
[0090] In summary, our findings provide a method of reducing HCC
growth with probiotic compositions via immunomodulation and open
new avenues to the development of more effective cancer
therapies.
[0091] It should be understood that the examples and embodiments
described herein are for illustrative purposes only and that
various modifications or changes in light thereof will be suggested
to persons skilled in the art and are to be included within the
spirit and purview of this application and the scope of the
appended claims. In addition, any elements or limitations of any
invention or embodiment thereof disclosed herein can be combined
with any and/or all other elements or limitations (individually or
in any combination) or any other invention or embodiment thereof
disclosed herein, and all such combinations are contemplated with
the scope of the invention without limitation thereto.
[0092] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
reference unless the context clearly dictates otherwise. Thus, for
example, reference to "a composition" includes a plurality of such
compositions, reference to "the composition" is a reference to one
or more compositions and equivalents thereof known to those skilled
in the art, and so forth.
[0093] Throughout the description and claims of this specification,
the word "comprise" and variations of the word, such as
"comprising" and "comprises," means "including but not limited to,"
and is not intended to exclude, for example, other additives,
components, integers or steps. "Optional" or "optionally" means
that the subsequently described event, circumstance, or material
may or may not occur or be present, and that the description
includes instances where the event, circumstance, or material
occurs or is present and instances where it does not occur or is
not present.
[0094] Ranges may be expressed herein as from "about" one
particular value, and/or to "about" another particular value. When
such a range is expressed, also specifically contemplated and
considered disclosed is the range from the one particular value
and/or to the other particular value unless the context
specifically indicates otherwise. Similarly, when values are
expressed as approximations, by use of the antecedent "about," it
will be understood that the particular value forms another,
specifically contemplated embodiment that should be considered
disclosed unless the context specifically indicates otherwise. It
will be further understood that the endpoints of each of the ranges
are significant both in relation to the other end-point, and
independently of the other endpoint unless the context specifically
indicates otherwise. Finally, it should be understood that all of
the individual values and sub-ranges of values contained within an
explicitly disclosed range are also specifically contemplated and
should be considered disclosed unless the context specifically
indicates otherwise. The foregoing applies regardless of whether in
particular cases some or all of these embodiments are explicitly
disclosed.
[0095] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
skill in the art to which the disclosed method and compositions
belong. Although any methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
the present method and compositions, the particularly useful
methods, devices, and materials are as described. Publications
cited herein and the material for which they are cited are hereby
specifically incorporated by reference. Nothing herein is to be
construed as an admission that the present invention is not
entitled to antedate such disclosure by virtue of prior invention.
No admission is made that any reference constitutes prior art. The
discussion of references states what their authors assert, and
applicants reserve the right to challenge the accuracy and
pertinency of the cited documents. It will be clearly understood
that, although a number of publications are referred to herein,
such reference does not constitute an admission that any of these
documents forms part of the common general knowledge in the
art.
[0096] Although the description of materials, compositions,
components, steps, techniques, etc. may include numerous options
and alternatives, this should not be construed as, and is not an
admission that, such options and alternatives are equivalent to
each other or, in particular, are obvious alternatives. Thus, for
example, a list of different bacteria does not indicate that the
listed bacteria are obvious one to the other, nor is it an
admission of equivalence or obviousness.
[0097] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the method and
compositions described herein. Such equivalents are intended to be
encompassed by the following claims.
* * * * *